The accurate immunological determination of proteins calls for antibodies that have a sufficiently high affinity to these proteins to allow them to be detected in the small amounts in which they are generally encountered. Furthermore, these antibodies must also be sufficiently specific to rule out any interference by other constituents of the medium in which the determination is carried out. However, such antibodies are often unavailable, because it is difficult to obtain a sufficiently large and suitably pure source of the antigen in question.
However, the use of synthetic peptides as immunogens has made it possible to obtain antibodies to antigens that are rare, to antigens of which only the amino acid sequence is known, and to certain weakly immunogenic epitopes located in a protein molecule. In most cases, however, the resulting antibodies to such synthetic peptides do not recognize the whole protein with a sufficient avidity to permit an immunoassay having the required sensitivity to be based on them.
Numerous attempts have been made to eliminate interference by the other constituents of the medium in which the protein immunoassay is conducted. In one of these methods, described in European Patent Application No. 51,985, the determination is carried out after the sample has been treated with a proteolytic agent. The way it is described there, this method permits the selective destruction of the epitopes on the contaminating proteins while leaving intact the epitopes on the protein to be determined, also permitting the determination of the protein fragments that carry specific epitopes and which are obtained by proteolytic cleavage. This is achieved there with the aid of antibodies obtained by immunizing animals with purified fragments obtained by cleaving the said protein.
However, both the techniques proposed in the publication mentioned above have some drawbacks. In the first technique outlined above, the selective destruction of the epitopes on the interfering protein molecules can only be accomplished if one knows exactly both a) the location of the epitopes that are recognized by the antibodies involved in the determination and b) the nature of the contaminating proteins, or if one carries out an extensive study of the different techniques of proteolytic fragmentation.
In the second technique outlined above, the determination of the proteolytically obtained fragments with the aid of antibodies obtained by immunization with purified fragments necessitates a large amount of very pure native protein. Furthermore, the detection of specific antibodies calls for the characterization of the proteolytically obtained purified fragments prior to their use as immunogens, or for a thorough investigation of the specificity of the antibodies obtained after immunization with the different peptides.
A method has also been described for the determination of the precursor of vasopressin by means of antibodies to vasopressin itself. In this method, the antigenic determinant is reconstituted prior to the determination, by means of an enzymatic and chemical treatment [see A. Cupo, G. Rougon-Rapuzzi and M. Delaage: "Immunochemical detection of vasopressin precursors: artificial processing and quantitation along the hypothalamo-hypophyseal axis", Eur. J. Biochem., 115 (1981) pp. 169-174].
A similar method has been used successfully to determine met-enkephalin by employing antibodies raised to a natural fragment of its precursor. This determination is carried out after treating the sample first with trichloroacetic acid in order to eliminate the natural fragment and then with trypsin [see A. Cupo, P. A. Pontarotti, T. Jarry and M. A. Delaage: "A new immunological approach to the detection and quantitation of met-enkephalin precursors in the rat brain", Neuropeptides, 4 (1984) pp. 375-387].